Mutant RBM20 hiPSC-derived cardiomyocytes carrying variants in the glutamate rich domain reveal aberrant splicing but normal intracellular localisation of the protein

Abstract Introduction Mutations in the RNA binding motif protein 20 (RBM20) encoded by the gene RBM20 are associated with severe and early onset forms of dilated cardiomyopathy (DCM). RBM20 acts as a splicing factor of several cardiac genes among them TTN and RYR2. Most of the disease-associated RBM20 mutations are localised in the highly conserved RSRSP-stretch within the arginine and serine rich domain (RS-domain) of the protein. Mutations in this domain lead to mislocalisation of RBM20 from the nucleus to the cytoplasm in DCM-myocardium and are associated with missplicing of RBM20 target genes. We and others identified in patients additional disease-associated mutations in the glutamate-rich domain of RBM20 (GRD). Mutations in GRD do not lead to mislocalisation of the protein in human myocardium but are nevertheless associated with aberrant splicing of RBM20 target genes. This suggests a different pathomechanism as compared to RS-domain variants. However, the exact pathomechanisms of GRD-mutations remain unclear. Purpose Goal of this study was to establish RBM20-p.V914A human induced pluripotent cell (hiPSC)-lines associated with DCM. These cells provide a human model system to characterise the pathomechanisms of RBM20 GRD-variants. Methods We used a nearly PAM-less base editor to introduce the variant RBM20-p.V914A in hiPSCs. The hiPSCs were differentiated and matured to hiPSC-derived cardiomyocytes (hiPSC-CMs). hiPSC-CMs were used i) for splicing analysis of RBM20 target genes by quantitative real-time PCR and ii) for immunofluorescence subcellular detection of RBM20 localisation. Results We generated heterozygous and homozygous hiPSC-lines with the pathogenic RBM20 variant p.V914A. Splicing analyses of the mutant hiPSC-CM confirmed aberrant splicing of TTN- and RYR2-transcripts compared to the wildtype. Immunofluorescence staining proved RBM20 p.V914A-localisation like the wildtype in the nucleus in both heterozygous and homozygous lines. Conclusion We generated two model cell lines for the analysis of mutations in the GRD of RBM20 which show splicing and localisation pattern which is comparable to the patterns in the patient's myocardium published previously. These findings underline that the pathomechanism of RGD mutations is different from mutations in the RSRSP-stretch. The presented cells will provide a crucial platform for dissecting the pathomechanisms of RBM20 mutations in the GRD in future studies. Additionally, they offer a valuable in vitro tool for the establishment of therapeutic gene-editing approaches.